I= PURIFICATION AND CHARACTERIZATION OF MICROBIAL CELLULOLYTIC ENZYMES
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Title of Thesis
PURIFICATION AND CHARACTERIZATION OF MICROBIAL CELLULOLYTIC ENZYMES

Author(s)
Amtul Jameel Sami
Institute/University/Department Details
Institute of Chemistry/ University of the Punjab
Session
1989
Subject
Chemistry
Number of Pages
236
Keywords (Extracted from title, table of contents and abstract of thesis)
cellulolytic enzymes, avicelase activities, mesophilic, cellulolytic, facultatively aerobic bacterium, cellulomonas flavigena

Abstract
A locally isolated, mesophilic, cellulolytic, facultatively aerobic bacterium identified as cellulomonas flavigena was studied for the production, purification and characterization of extracellular cellulolytic enzymes. When the microbe was cultivated in the presence of 0.5% Avicel as carbon source, it produced higher levels of CMCase as well as avicelase activities as compared to those produced in the presence of 0.5% CMC. Tween-80 when added to the culture ,medium, at a concentration pf 0.1% resulted in more than two fold increase in the enzyme activities in the presence of Avicel as substrate. Extra cellular CMCase and avicelase activities increased with the increase in concentration of yeast extract upto a level of 0.2%, beyond which no further increase took place in the enzyme activities. Maximal CMCase and avicelase activities were 10.0 and 1.2 U/m1 of the culture supernatant. These were obtained after 72 h of fermentation at which stage, the growth as determined in terms of cellular proteins, was maximal. Production of extracellular cellulases, both free and Avicel bound was studied in the presence of different concentrations of Avicel. It was found that both the enzyme activities in the culture supernatant were maximum in the presence of 0.5% Avicel. Beyond this concentration there was a decrease in the enzyme activities in the culture supernatant.

Characteristics of Avicelase and CMCase activities were studied and it was found that Avicelase and CMCase were most active at pH 6.5 and 500C. end product of each of the enzyme activity was carbohydrates. Both the enzyme activities were stable in the Ph range of 3-11 and 0-100C and 100% activities were lost when incubated at 700C for 30 minutes. Calcium and magnesium activated the enzymes when used in the concentration of 10-15mM, while NaCl had no effect on both the activities. Heavy metal ions i.e. silver and iron inhibited both the enzymes. Enzymes were inhibited in the presence of cellobiose and glucose upto 30mM in the reaction mixture. B-mercaptoethanol strictly inactivated the enzyme. C. flavingena also secreted protease activity which showed two Ph optima i.e. 6.5 and 8.9.

A substantial amount of cellulase activity bound to the residual avicel during fermentation. Conditions were optimized for the elution of the substrate bound cellulases from residual Avicel. Sonication or shaking of residual Avicel from the specific hours of fermentation medium with distilled water were equally effective for the elution of substrate-bound enzymes. Maximum CMCase activities were bound to the substrate during initial stages of fermentation. Higher proportions of avicelase activity remained bound to be insoluble cellulosic substrate throughout the fermentation periods studied. Maximal total CMCase activities (both the free and substrate bound activities) obtained with 2% Avicel as carbon source were 13U/ml and for avicelase it was 3.0 U/ml of the culture medium.

A comparative study of substrate bound and free CMCase activities was done on PACE followed by zymogram technique. It was found that free activities comprised of at least 7 CMCase bands while the bound activities showed only four major bands active against CMC.

For purification of CMCases a sequential procedure was employed including precipitation with 80% acetone, gel filtration, in-exchange chromatography and preparative gradient PAGE. First two purification steps increased specific activities upto three times e.g . 330 U CMCase/mg protein and 36 U avicelase/mg protein. Each of the purified enzyme CMCase 1, CMCase 2, CMCase 3, CMCase 4, and CMCase 5 showed single band on native PAGE and SDS-PAGE.

Characteristics of all the purified CMCase were studied and it was found that all of them were active at 45-500C and were only active against CMC. The major hydrolytic products of all the enzymes were ooligosaccharides and cellobiose. Each of the enzyme was inhibited in the presence of cellobiose and glucose when added to above 15Mm concentration. Enzymes were stable in the temperature range of 0 - 100C and Ph of 4 €“ 9.5. Ca++ Mg++ has no considerable effect on the enzyme activities while iron and silver were proved to be their strict inhibitors. B-mercepto ethanol also inhibited these enzymes. The purified enzymes differed in their motilities on non denaturing gradient PAGE as appeared on zymogram. PH optima of these enzymes ranged between 6.5€“7.0 CMCase 1 showed a sharp PH optimum at 7.0 and CMCase 4 exhibited maximum activities at PH 6.5. molecular weights of each of the enzymes were estimated by SDS-PAGE. CMCase 1 and CMCase 2 were both found to have molecular weight of 20,400, CMCase 3 and 5 had same molecular weight of 80,000. km value of each of the purified enzyme was determined y using CMC as substrate. These were 0.83,1.7, 1.54, 1.64, 1.6 g/1 for CMCase 1,2,3,4 and 5 respectively.

Download Full Thesis
1369.2 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
67.08 KB
2 1 Introduction 1
241.72 KB
  1.1 Cellulose 1
  1.2 Hydrolysis 1
  1.3 Cellulases 6
  1.4 Adsorption 7
  1.5 Active Sites 10
  1.6 Mode Of Action 11
  1.7 Kinetic Approach 13
  1.8 Enzymological Approach 16
  1.9 Production 20
  1.10 Purification And Characterization 20
  1.11 Genetic Engineering 28
  1.12 Applications Of Cellulases 31
3 2 Materials And Methods 33
88.88 KB
  2.1 Microorganism 33
  2.3 Culture Methods 33
  2.4 Determination Of Growth Rate 34
  2.5 Production 34
  2.6 Isolation Of Enzymes 35
  2.7 Protein Estimation 35
  2.8 Enzyme Assays 36
  2.9 Effect Of pH, Temperature And Inhibition Studies 37
  2.10 Effect Of B-Mercaptoethanol 37
  2.11 Effect Of Metal Ions 37
  2.12 Stability Of Cellulases 37
  2.13 Substrate Specificity 38
  2.14 Effect Of Substrate Concentration 38
  2.15 Identification Of Hydrolytic Product 38
  2.16 Purification Of Cellulases 39
  2.17 Acetone Precipitation 39
  2.18 Gel Filtration 39
  2.19 Ion-Exchange Chromatography 40
  2.20 Electrophoresis 40
  2.21 Detection Of Proteins 41
  2.22 Identification Of CMCase Bands 41
  2.23 Elution Of Proteins From The Gel 42
  2.24 Molecular Weight Determination 42
4 3 Results 43
439.39 KB
  3.1 Purification 56
  3.2 Characteristics 62
  3.3 Purification Of Cellulases 73
5 4 Discussion 119
86.92 KB
6 5 References 130
421.9 KB
  5.1 Abbreviation
  5.2 List Of Publication S